The present invention relates to specific binding members, particularly antibodies and fragments thereof, which bind to the necrotic centres of tumours. These specific binding members are useful in the treatment of cancer.
The treatment of proliferative disease, particularly cancer, by chemotherapeutic means often relies upon exploiting differences in target proliferating cells and other normal cells in the human or animal body. For example, many chemical agents are designed to be taken up by rapidly replicating DNA so that the process of DNA replication and cell division is disrupted. Another approach is to identify antigens on the surface of tumour cells which are not normally expressed in developed human tissue, such as embryonic antigens. Such antigens can be targeted with binding proteins such as antibodies to deliver a toxic agent to or other substance which is capable directly or indirectly of activating a toxic agent at, the site of a tumour.
An alternative approach which has been developed more recently relies on the fact that a significant proportion of tumour cells are in various stages of cell degeneration and death. Unlike programmed cell death (apoptosis) which occurs during the natural turnover of certain cell types, tumour cells undergo a degenerative and less controlled death during which they have been found to exhibit abnormal surface membrane permeability. European patent application EP-A-270340 describes the preparation of murine antibodies to nuclear components of cells which are able to target necrotic cells in a tumour by exploiting this phenomenon. Miller et al (Hybridoma, 1993, 12, 689-697) describe a particular antibody prepared in accordance with EP-A-270340. This antibody, TNT-1, was found to bind histone fraction H1. The histones are the most abundant proteins in chromatin, the concentration of each type of histone being approximately 5000 times higher than the concentration of a typical sequence-specific DNA-binding protein. Epstein et al (in xe2x80x9cHandbook of Targeted Delivery of Imaging Agentsxe2x80x9d CRC press, Inc., 1995, ed. VP Torchilin) describes the uptake of radiolabelled TNT-1 into nude mice bearing the ME-180 cervical carcinoma, and report that there was no appreciable labelling of other organs. The authors also used the F(abxe2x80x2)2 fragment of TNT-1 labelled with 131I for tumour imaging studies in human patients.
A known problem with the use of murine antibodies in human therapy is that repeat treatment of such antibodies leads to a human anti-mouse antibody (HAMA) response. HAMA responses have a range of effects, from neutralisation of the administered antibody leading to a reduced therapeutic dose, through to allergic responses, serum sickness and renal impairment. In order to overcome these disadvantages humanization of antibodies has been developed. More recently, repertoires of human antibodies have been cloned and these can be screened, for example by phage display technology, (McCafferty et al, WO92/01047) to identify human antibodies specific for human antigens.
We have investigated the binding properties of the TNT-1 antibody and attempted to secure human antibodies with similar binding profiles and useful tumour localisation in animal models. We have found that although TNT-1 binds primarily to nuclear histone H1 it also binds a complex pattern of other histones and non-identified proteins in a nuclear preparation. We were able to identify a number of human antibodies, screened primarily against a nuclear extract and further against histone H1 which had similar binding profiles. Despite the similarities in binding profiles between the human antibody clones analysed, only one (identified below as xe2x80x9cNHS76xe2x80x9d) was found to additionally exhibit low cross reactivity to non-necrotic tissues and organs and good tumour:blood localisation ratio. Other candidate antibodies tested either showed cross-reactivity or had poorer tumour:blood ratios of localisation.
Accordingly, specific binding proteins such as antibodies which are based on the complementarity-determining regions (CDRs) of the NHS76 antibody identified, particularly the CDR 3 regions, will be useful for targeting the necrotic centres of malignant tumours.
In the accompanying drawings, the nucleic acid sequence and translation thereof of the NHS76 VH gene is shown in FIG. 1. The nucleic acid sequence is designated SEQ ID NO:1. The translation is SEQ ID NO:2. The VL gene of NHS 76 is shown as FIG. 2. Its nucleic acid sequence is designated SEQ ID NO:3, and its translation as SEQ ID NO:4. In both figures, the CDR""s are indicated in boxes.
In a first aspect the present invention provides an isolated specific binding member capable of binding an intracellular antigen, wherein said specific binding member comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out as residues 99 to 106 of SEQ ID NO:2. The invention further provides said isolated specific binding member which further comprises one or both of the polypeptide binding domains substantially as set out as residues 31-36 and 51-66 of SEQ ID NO:2, preferably both. In a preferred embodiment, the binding domains are carried by a human antibody framework. One example of such an embodiment is the sequence substantially as shown in SEQ ID NO:2.
In a second aspect, the invention provides an isolated specific binding member capable of binding an intracellular antigen, wherein said specific binding member comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out as residues 88 to 98 of SEQ ID NO:4. The invention further provides said isolated specific binding member which further comprises one or both of the polypeptide binding domains substantially as set out as residues 23-33 and 49-55 of SEQ ID NO:4, preferably both. In a preferred embodiment, the binding domains are carried by a human antibody framework. One example of such an embodiment is the sequence substantially as shown in SEQ ID NO:4.
In a particularly preferred embodiment, the invention provides a specific binding member which comprises a first specific binding member comprising a sequence substantially as set out as residues 99 to 106 of SEQ ID NO:2 in association with a second specific binding member comprising a sequence substantially set out as residues 88 to 98 of SEQ ID NO:4. Such a specific binding member according to the invention may be in the form of an antibody F(abxe2x80x2)2 fragment.
Specific binding members of the invention may carry a detectable or functional label.
In further aspects, the invention provides an isolated nucleic acid which comprises a sequence encoding a specific binding member as defined above, and methods of preparing specific binding members of the invention which comprise expressing said nucleic acids under conditions to bring about expression of said binding member, and recovering the binding member.
Specific binding members according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment of a tumour in a human patient which comprises administering to said patient an effective amount of a specific binding member of the invention.
These and other aspects of the invention are described in further detail below.